Use of cysteine derivatives for the preparation of a medicament intended to treat pathologies which result from the formation of the heterotrimeric G protein

ABSTRACT

The invention relates to the use of cysteine derivatives for preparing a medicament intended to treat diseases which result from the formation of the heterotrimeric G protein. These diseases include in particular diseases linked to the following biological functions or disorders: smell, taste, perception of the light, neurotransmission, neurodegeneration, endocrine and exocrine gland functions, autocrine and paracrine regulation, arterial tension, embryogenesis, benign cell proliferation, oncogenesis, viral infection, immunological functions, diabetes, obesity, and benign :and malign proliferative diseases. Said cysteine derivatives include in particular: bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine disulphide (I), and bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphtyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl) disulphide (II).

The present invention relates in particular to the use of derivatives ofcysteine for the preparation of a medicament intended to treatpathologies which result from the formation of the heterotrimeric Gprotein. These diseases include in particular diseases linked to thefollowing biological functions or disorders: smell, taste, perception oflight, neurotransmission, neurodegeneration, endocrine and exocrinegland functions, autocrine and paracrine regulation, arterial tension,embryogenesis, benign cell proliferation, oncogenesis, viral infection,immunological functions, diabetes, obesity, and benign and malignproliferative diseases.

The G proteins are in fact the structural association of three distinctsub-units called α, β, and γ, but operate as dissociable entitiesconstituted by α sub-units on the one hand and β/γ dimers on the otherhand.

The G proteins participate in the transmission of signals outside thecell thanks to its interaction with receptors with seven transmembranedomains inside using different effectors including adenylate cyclase,phospholipase C or also the ionic channels. The adenylate cyclase enzymegenerates cyclic AMP (cAMP) (cf. Gilman, A. G. Biosci. Rep. 15, 65-97(1995)). Thus, it is known that, in order to activate the adenylatecyclase, it is necessary for the G proteins to be transitionally in aheterotrimeric form, in which form the monomer constituted by an αsub-unit is associated with the dimer constituted by the β and γsub-units. It is only in this situation that the signal outside the cellcan activate the α sub-unit of a G protein, which can, afterdisassociation, modulate the adenylate cyclase and modulate theproduction of cAMP.

It is also known that the β/γ dimers can directly activate the effectorsleading to the activation of kinases regulated by extracellular signals(ERKs) or MAP kinases. A direct link between the β/γ sub-units and thesrc or src like kinases has been demonstrated (cf. Gutkind, J. S. J.Biol. Chem. 273, 1839-1842 (1998)).

Moreover, bacterial toxins such as Vibrio cholera and Bortellapertussis, peptides such as mastoparan and suramin have been presentedas directly modulating the activity of the G proteins (cf. Freissmuth,M., Boehm, S., Beindl, W., et al. Mol. Pharmacol. 49, 602-611 (1996);Boehm, S., Huck, S., Motejlek, A., et al. Journal of Neurochemistry 66,1019-1026 (1996); Cachero, T. G., Rigual, R., Rocher, A. & Gonzalez, C.Eur. J. Neurosci. 8, 2320-2327 (1996); Danilenko, M., Worland, P.,Carlson, B., Sausville, E. A. & Sharoni, Y. Biochem. Biophys. Res.Commun. 196, 1296-1302 (1993); Beindl, W., Mitterauer, T., Hohenegger,M., Ijzerman, A. P., Nanoff, C. & Freissmuth, M. Mol. Pharmacol. 50,415-423 (1996)).

For example, the choleric toxin modifies the as sub-unit of the Gprotein by adding an ADP-ribose originating from the NAD to anarginine-specific acceptor site. This completely blocks the activity ofthe GTPase, provoking persistent stimulation of its next effector,adenylate cyclase and leading to overproduction of cAMP.

The harmful effects of an abnormal cAMP level are also known and occurin particular at the level of the following biological functions ordisorders: smell, taste, perception of light, neurotransmission,neurodegeneration, endocrine and exocrine gland functions, autocrine andparacrine regulation, arterial tension, embryogenesis, benign cellproliferation, oncogenesis, viral infection and immunological functions,diabetes and obesity.

The Applicant has just discovered that certain derivatives of cysteine,namely the compounds of general formula (A)

corresponding to sub-formulae (A1) or (A2):

in which:

X represents R₁₂ and Y represents R₈, or X and Y complete a ring with 6members, the X-Y set representing the —CH(R₈)—CH(R₉)-radical;

R₁ represents H, a lower alkyl or alkylthio radical;

R₂ and R₃ represent independently H or a lower alkyl radical;

R₄ represents H₂ or O;

R₅ represents H, or one of the lower alkyl, lower alkenyl, loweralkynyl, aryl, lower arylalkyl, heterocycle or lower alkyl heterocycleradicals, these radicals being optionally substituted by radicals chosenfrom the group comprising a lower alkyl radical, —O—R₁₀, —S(O)_(m)R₁₀ (mrepresenting 0, 1, or 2), —N(R₁₀)(R₁₁), —N—C(O)—R₁₀, —NH—(SO₂)—R₁₀,—CO₂—R₁₀, C(O)—N(R₁₀)(R₁₁), and —(SO₂)—N(R₁₀)(R₁₁);

R₆ and R₇ represent independently H, a —C(O)—NH—CHR₁₃—CO₂R₁₄ radical, orone of the lower alkyl, aryl, lower arylalkyl, heterocycle or loweralkyl heterocycle radicals, these radicals being optionally substitutedby radicals chosen from the group comprising the OH, alkyl or loweralkoxy, N(R₁₀)(R₁₁), COOH, CON(R₁₀)(R₁₁), and halo radicals,

or R₆ and R₇ form together an aryl radical or a heterocycle;

R₈ and R₉ represent independently, H, or one of the lower alkyl, aryl,lower arylalkyl, heterocycle or lower alkyl heterocycle radicals, theseradicals being optionally substituted by radicals chosen from the groupcomprising the OH, alkyl or lower alkoxy, N(R₁₀)(R₁₁), COOH,CON(R₁₀)(R₁₁) and halo radicals,

or R₈ and R₉ together form an aryl radical or a heterocycle;

R₁₀ and R₁₁ represent independently H, an aryl radical or a heterocycle,or an alkyl, arylalkyl or lower alkyl heterocycle radical;

R₁₂ represents NR₉, S, or O;

R₁₃ represents a lower alkyl radical optionally substituted by a radicalchosen from the lower alkyl, —OR₁₀, S(O)_(m)R₁₀ (m representing 0, 1, or2) and —N(R₁₀)(R₁₁) radicals;

R₁₄ represents H or a lower alkyl radical;

or the compounds of general formula (B):W₁—Ar—W₂   (B)in which:

W₁ represents a remainder originating from a cysteine in reduced or nonreduced form;

Ar represents a radical derived from an aminobenzoic acid, the aromaticring of which is optionally substituted;

W₂ represents an amino acid, preferably an aliphatic amino acid;or also the compounds of general formula (C):

in which:

Z₁ represents a lower alkyl radical;

Z₂ and Z₃ both represent H or Z₂ and Z₃ together form a chain having 2to 4 elements chosen from the —C(O)—, —CH₂—, —CH(NH₂)— and —S-radicals,it being understood that two successive elements are not both —C(O)—;

it being understood that the compounds of general formula (C) can alsobe presented in the form of dimers, when the Z₂ radical represents ahydrogen atom which can be eliminated by oxidization;

or also a pharmaceutically acceptable salt of a compound of generalformula (A), (B) or (C);

can be used to prepare medicaments intended to treat pathologies whichresult from the formation of the heterotrimeric G protein.

By lower alkyl radical, is understood a linear or branched alkyl radicalcontaining 1 to 6 carbon atoms, and in particular the methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl, pentyl,neopentyl, isopentyl, hexyl, isohexyl radicals. By heterocycle radicalis understood a radical constituted by one or more rings and includingat least one heteroatom. By arylalkyl, alkyl heterocycle, alkylthio orlower alkoxy radical, is understood the radicals of which the alkylradical has the meaning indicated previously.

Preferably, the Ar radical included in formula (B) is optionallysubstituted by an alkyl radical comprising 1 to 6 carbon atoms or anaryl radical, these alkyl or aryl radicals themselves being optionallysubstituted preferentially by an alkoxy radical having 1 to 4 carbonatoms, fluoro, chloro, bromo. The aryl radical preferably a phenyl canitself be substituted by an alkyl radical.

Preferably also, the compounds of general formula (B) are such that Arrepresents a radical derived from an aminobenzoic acid the aromatic ringof which is substituted by a phenyl radical and W₂ represents analiphatic amino acid.

In particular, the following compounds can be used to preparemedicaments intended to treat pathologies which result from theformation of the heterotrimeric G protein:

-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piperazine;-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]disulphide;-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide;-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)    disulphide;-   the compound of formula:-   the compound of formula:-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,    or its dimeric form;-   and    7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine;    or also a pharmaceutically acceptable salt of one of these    compounds.

One of the following compounds is preferably used for the invention:

-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide (I);-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)    disulphide (II);-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    (III);-   the compound of formula:-   7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    (V);-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine]    disulphide (VI);-   the compound of formula:-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   1-[2(R)-amino-3-mercaptopropyl]-2(S)-n-butyl-4-(1-naphthoyl)piperazine.    or a pharmaceutically acceptable salt of one of the latter.

More preferentially, one of the following compounds is used for theinvention:

-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide (I);-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)    disulphide (II);-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    (III);-   7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo(1.2a]pyrazine    (V);-   the compound of formula:    or a pharmaceutically acceptable salt of one of the latter.

Finally, the following compounds are more particularly preferred:

-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide (I);-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)    disulphide (II);    or a pharmaceutically acceptable salt of one of the latter.

The invention therefore relates firstly to the use of the compounds ofgeneral formula (A), (B) or (C) as described previously for preparing amedicament intended to treat pathologies which result from the formationof the heterotrimeric G protein. In particular, it relates to the use ofsaid inhibitors for preparing medicaments intended to treat diseaseslinked to the following biological functions or disorders: smell, taste,perception of light, neurotransmission, neurodegeneration, endocrine andexocrine gland functions, autocrine and paracrine regulation, arterialtension, embryogenesis, viral infection, immunological functions,diabetes and obesity.

More particularly, the invention relates to the use of compounds ofgeneral formula (A), (B) or (C) for preparing a medicament intended totreat cholera, Acquired Immune Deficiency Syndrome (AIDS), traveldiarrhea and familial masculine precocious puberty.

A subject of the invention is also new products of general formula (A)numbered 1 to 7 and described hereafter in the examples, namely:

-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,    or its dimeric form;-   and    7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine.

A subject of the invention is also said new products or theirpharmaceutically acceptable salts as medicaments, as well as their usefor preparing a medicament intended to treat pathologies which resultfrom the formation of the heterotrimeric G protein. In particular, itrelates to the use of said products for preparing medicaments intendedto treat diseases linked to the following biological functions ordisorders: smell, taste, perception of light, neurotransmission,neurodegeneration, endocrine and exocrine gland functions, autocrine andparacrine regulation, arterial tension, embryogenesis, benign cellproliferation, oncogenesis, viral infection, immunological functions,diabetes, obesity, and benign and malign proliferative diseases.

The products particularly preferred for use according to the inventionare therefore the following:

-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide;-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)    disulphide;-   the compound of formula:-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;-   7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methyoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,    or its dimeric form;-   and    7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;    or a pharmaceutically acceptable salt of one of the latter.

Similarly, the invention relates more particularly to the use of thecompounds previously mentioned for preparing a medicament intended totreat cholera, Acquired Immune Deficiency Syndrome (AIDS), traveldiarrhea and familial masculine precocious puberty.

The compounds of general formula (A) and their preparation are describedin the Patent Application WO 97/30053 or in the examples hereafter. Thecompounds of general formula (B) and their preparation are described inthe Patent Application WO 96/21456. Finally, the preparation of thecompounds of general formula (C) is described in the Patent ApplicationPCT WO 95/00497, except for the compound of formula (VII) for which thesynthesis is described in the experimental part of this Application.

The pharmaceutical compositions comprising a compound of the inventioncan be in the form of solids, for example powders, granules, tablets,gelatin capsules, liposomes or suppositories. The appropriate solidsupports can be, for example, calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine and wax.

The pharmaceutical compositions comprising a compound of the inventioncan also be presented in liquid form, for example, solutions, emulsions,suspensions or syrups. The appropriate liquid supports can be, forexample, water, organic solvents such as glycerol or glycols, as well astheir mixtures, in varying proportions, in water.

The administration of a medicament according to the invention can becarried out by topical, oral, parenteral route, by injection(intramuscular, sub-cutaneous, intravenous, etc.), etc. Theadministration route will of course depend on the type of disease to betreated.

The administration dose envisaged for a medicament according to theinvention is comprised between 0.1 mg and 10 g depending on the type ofpathology to be treated.

Unless they are defined in another manner, all the technical andscientific terms used here have the same meaning as that usuallyunderstood by an ordinary specialist in the field to which thisinvention belongs. Similarly, all the publications, patent applications,all the patents and all other references mentioned here are incorporatedby way of reference.

EXAMPLES Example 17-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:1

Compound 1 was prepared according to the synthesis diagram below:

1.a) Carbobenzyloxy-L-cyclohexylalanine

L-phenylalanine (10.0 g; 60.6 mmol) is combined with PtO2 (430 mg) inacetic acid (60 ml) and the mixture is hydrogenated overnight under20-50 psi H₂. A 5% aqueous solution of HCl is added to the mixture inorder to obtain a limpid solution and hydrogenation is continued untilthe consumption of hydrogen ceases. The catalyst is eliminated byfiltration and the filtrate is concentrated under reduced pressure. Theresidue is taken up in methanol and water and the pH is adjusted to 4.4by the addition of a 10% solution of NaOH. The product obtained isrecovered by filtration and used without further purification.

L-cyclohexylalanine (60.6 mmol) is suspended in water (100 ml), K₂CO₃(8.36 g; 60.6 mmol) is added, then a solution ofN-(benzyloxycarbonyloxy)succinimide (15.1 g; 60.6 mmol) in CH₃CN (150ml) and the mixture obtained is agitated vigorously for 45 minutes. Themixture is concentrated in order to produce a volume of approximately100 ml and washed with Et₂O (100 ml), then acidified with concentratedHCl and extracted with AcOEt (2×50 ml). The combined AcOEt phases aredried over Na₂SO₄, filtered and concentrated in order to produce a clearoil (17.27 g; 93%). NMR ¹H (DMSO-d6): 7.5-7.6 (1H, d); 7.2-7.5 (5H, m);5.0-5.1 (2H, s); 3.9-4.1 (1H, m); 0.7-1.8 (13H, m).

1.b)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-(cyclohexyl)methyl)-4-(2-methylphenyl)-imidazole

Cbz-(L)-cyclohexylalanine (4.58 g; 15.0 mmol) and Cs₂CO₃ (2.44 g; 7.50mmol) are placed in a 2:1 mixture of DMF:H₂O (75 ml). The mixtureobtained is agitated until it becomes homogeneous. The solvents areeliminated under reduced pressure, the residue is dissolved in DMF (60ml) and 2-bromo-2′-methylacetophenone (3.20 g; 15.0 mmol) in DMF (30 ml)is added. The mixture is agitated overnight at ambient temperature thenfiltered and concentrated under reduced pressure. The keto-esterobtained is solubilized in xylenes (100 ml) and ammonium acetate (19.5g; 0.25 mol) is added. The mixture is heated to reflux for approximately3 hours with elimination of the excess AcONH₄ supernatant and of thewater released by means of a Dean-Stark trap. The reaction mixture isconcentrated under reduced pressure, taken up in AcOEt and washed with asaturated solution of NaHCO₃ (100 ml) and with a saturated solution ofNaCl (100 ml). The AcOEt phase is dried over Na₂SO₄, filtered andconcentrated under vacuum. The crude product obtained is purified byflash chromatography on silica gel with a CHCl₃/MeOH mixture 98/2 aseluant. The fractions containing the pure product are combined andconcentrated in order to produce the product (2.52 g; 40%) in the formof a slightly brown foam which is used in the following stage withoutadditional purification.

1.c)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-(cyclohexyl)methyl)-1-((2-ethoxy-2-oxo)ethyl)-4-(2-methylphenyl)-imidazole

Intermediate 1.b (2.52 g; 6.0 mmol) is solubilized in DMF (20 ml) andtreated with K₂CO₃ (1.67 g, 12.1 mmol) and ethyl bromoacetate (1.34 ml;12.5 mmol) is added. The mixture obtained is heated at 45° C. for oneand a half hours. The mixture is diluted with ether (50 ml) and washedwith a saturated solution of NaHCO₃ solution (50 ml) then with asaturated solution of NaCl (50 ml). The ethereal layer is dried overNa₂SO₄, filtered and concentrated in order to produce an oil which isused in the following stage without additional purification.

Mass spectrum: 504.3 MH+.

1.d)8-(cyclohexylmethyl)-6-oxo-2-(2-methylphenyl)-imidazo[1,2-a]pyrazine

The crude intermediate of Stage 1.c is solubilized in acetic acid (50ml) containing a 10% Pd on carbon catalyst (152 mg), then hydrogenatedunder a pressure of 50 psi of H₂ for 18 hours at ambient temperature.The catalyst is eliminated by filtration and the filtrate is heated at70° C. for 2 hours. The mixture obtained is concentrated under reducedpressure, dissolved in CH₂Cl₂ (100 ml) and washed with a saturatedsolution of NaHCO₃ (100 ml). The CH₂Cl₂ layer is dried over Na₂SO₄,filtered and concentrated in order to produce a viscous oil which isused in the following stage without additional purification.

Mass spectrum: 324.3 MH+.

1.e)8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo[1,2-a]pyrazine

The crude intermediate of Stage 1.c is solubilized in THF (25 ml) andtreated at ambient temperature with a 1M solution of BH3 in THF (25 ml)for half an hour then taken to reflux for 1 hour. The mixture is cooleddown using an ice bath and 4N HCl (40 ml) is added dropwise at 0° C. Themixture is taken to ambient temperature then taken to reflux for 1 hour.The reaction medium is then cooled down, filtered and concentrated underreduced pressure. The residue is treated with a saturated solution ofNaHCO₃ (50 ml) and extracted with CH₂Cl₂ (3×50 ml). The CH₂Cl₂ phasesare dried over Na₂SO₄, filtered and concentrated in order to produce aslightly brown oil (1.63 g; yield of 87% relative to Stages 1.c, 1.d and1.e).

Mass-spectrum: 310.3 MH+.

1f.)8-(cyclohexylmethyl)-7-[2-(((1,1-dimethylethoxy)carbonyl)amino)-1-oxo-3-((triphenylmethyl)thio)propyl]-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine

Diisopropylcarbodiimide (908 μl; 5.80 mmol) and BocCys(trt)-OH (5.37 g;11.6 mmol) are solubilized in CH₂Cl₂ (25 ml), the mixture obtained beingagitated for 45 minutes.8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo[1,2-a]pyrazine(1.63 g; 5.27 mmol) is then added. The reaction mixture is agitatedovernight at ambient temperature. The solvent is eliminated underreduced pressure and the product obtained is purified by flashchromatography on silica gel with a CH₂Cl_(2/)MeOH mixture 98/2 aseluant. The pure fractions are concentrated in order to produce aviscous oil which is which is used in the following stage withoutadditional purification.

Mass spectrum: 755.6 MH+.

1.g)7-(2-amino-1-oxo-3-(mercaptopropyl))-8-(cyclohexylmethyl)-2-(2-methylphenyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine:1

The intermediate of Stage 1.f (3.54 g; 4.69 mmol) is solubilized intrifluoroacetic acid (TFA, 80 ml) containing triisopropylsilane (1.92ml; 9.38 mmol) and the reaction mixture is agitated at ambienttemperature under nitrogen for one hour. The reaction mixture isfiltered and the filtrate is concentrated under reduced pressure. Theresidue is extracted by trituration with an aqueous solution of TFA at0.1% (6×65 ml) and filtered. The crude product is purified bypreparative HPLC on a C18 column using a gradient of 0 to 20% of CH₃CNin an aqueous solution of TFA at 0.1% for 30 minutes. The pure fractionsof the product are collected and lyophilized. The initial product islyophilized twice from a dilute solution of HCl in order to produce theproduct in the form of its hydrochloride (740 mg; 32%).

Mass spectrum: 413.2 MH+. NMR ¹H (DMSO-d6): 8.5-9.0 (3H, d, broad);7.8-8.0 (1H, s); 7.5-7.7 (1H, d); 7.2-7.5 (3H, m); 5.8-6.1 (1H, m);4.65-4.8 (1H, s); 4.5-4.7 (1H, d); 4.1-4.4 (2H, m); 3.8-4.0 (1H, m);3.2-3.7 (H₂O); 2.8-3.1 (2H, m); 2.35-2.5 (3H, s); 2.0-2.2 (1H, m);1.8-2.05 (2H, m); 1.25-1.4 (4H, broad s); 1.3-0.9 (6H, m).

Example 27-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:2

Compound 2 is prepared according to diagram 1, Stages b to g, accordingto a method similar to that of Example 1, 2-bromoacetophenone replacing2-bromo-2′-methylacetophenone in Stage b.

Mass spectrum: 399.2 MH+. NMR ¹H (DMSO-d6): 8.5-8.9 (3H, broad d);8.0-8.2 (1H, s); 7.8-8.0 (2H, d); 7.45-7.56 (2H, t); 7.35-7.5 (1H, t);5.9-6.05 (1H, broad s); 4.65-4.8 (1H, s); 4.5-4.65 (1H, d); 4.1-4.35(2H, m); 3.8-4.0 (1H, m); 3.2-3.8 (H₂O); 3.25-3.4 (1H, t); 2.8-3.05 (2H,m); 2.05-2.2 (1H, d); 1.85-2.05 (2H, t); 1.55-1.75 (4H, broad s);1.15-1.3 (1H, broad s); 1.2-0.9 (5H, m).

Example 37-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:3

Compound 3 is prepared according to diagram 1, Stages b to g, accordingto a method similar to that of Example 1, Boc-(L)-Ser(Bzl)-OH replacingCbz-(L)-cyclohexylalanine in Stage b and Stage d being replaced by adeprotection using TFA and iPr₃SiH according to a method similar toreaction 1.g. The product is obtained in the form of a pair ofdiastereoisomers in a proportion of 2:3.

Mass spectrum: 453.2 MH+.

The retention times for the diastereoisomers are 6.58 and 7.07 minutesrespectively in the following HPLC system: Eluant: 30-50% CH₃CN/0.1% TFADuration of elution: 24 minutes Detection: 254 nm Column: Vydac proteinand C18 peptide

Example 47-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)ethyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:4

Compound 4 is prepared according to diagram 1, Stages b to g, accordingto a method similar to that of Example 3, Boc-(L)-Thr(Bzl)-OH replacingBoc-(L)-Ser(Bzl)-OH in Stage b.

Mass spectrum: 467.3 MH+. NMR ¹H (DMSO-d6): 8.5-8.9 (3H, d, broad);8.0-8.1 (1H, s); 7.95-8.1 (2H, d); 7.4-7.5 (1H, t); 7.15-7.3 (1H, d);7.0-7.2 (3H, m); 6.9-7.05 (2H, m); 5.85-5.95 (1H, d); 4.75-4.85 (1H,broad s); 4.65-4.8 (1H, broad s); 4.35-4.65 (3H, m); 4.1-4.25 (2H, q);3.9-4.0 (3H, s); 2.8-3.1 (2H, m); 1.2-1.4 (3H, d).

Example 57-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine:5

Compound 5 was prepared according to the following synthesis diagram:

5.b)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-(2-oxo-2-(phenylmethoxy)ethyl)-4-(2-methoxyphenyl)-imidazole

Cbz-(L)-Asp(Obzl)-OH (5.00 g; 14.0 mmol) and Cs₂CO₃ (2.28 g; 7.00 mmol)are mixed in a 1:1 mixture of DMF:H₂O (75 ml). The mixture obtained isagitated until it becomes homogenous. The solvents are eliminated underreduced pressure, the residue is dissolved in DMF (60 ml) and2-bromo-2′-methoxyacetophenone (3.21 g; 14.0 mmol) in DMF (30 ml) isadded. The mixture obtained is agitated for half an hour at ambienttemperature then filtered and concentrated under reduced pressure. Theketo-ester obtained is triturated with a 1:1 mixture of Et₂O:hexanes(2×40 ml) then suspended in xylenes (100 ml). Ammonium acetate (17.5 g;0.23 mol) is added and the mixture is heated at reflux for approximatelyone hour and 30 minutes with elimination of the excess AcONH₄ and of thewater released by means of a Dean-Stark trap. The reaction medium iswashed with a saturated solution of NaHCO₃ (50 ml), dried over Na₂SO₄,filtered and concentrated under vacuum in order to produce 6.66 g (98%)of desired product.

Mass spectrum: 486.3 (MH+).

5.i)2-(1-(S)-((phenylmethoxy)carbonyl)-amino-2-((2-phenylmethoxy-2-oxo)ethyl)-1-((2-ethoxy-2-oxo)ethyl)-4-(2-methoxyphenyl)-imidazole

Intermediate 5.i is prepared according to a method similar to that ofStage 1.c.

Mass spectrum: 572.3 MH+.

5.j)(6-oxo-2-(2-methoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazin-8-yl)-aceticacid

Intermediate 5.j is prepared according to a method similar to that ofStage 1.d.

Mass spectrum: 302.2 MH+. NMR ¹H (DMSO-d6): 8.35-8.5 (1H, d, broad);8.0-8.1 (1H, dd); 7.45-7.55 (1H, s); 7.15-7.25 (1H, m); 7.0-7.1 (1H, m);6.9-7.0 (1H, m); 4.85-5.0 (1H, broad s); 4.55-4.75 (2H, q); 3.85-3.95(3H, s); 2.8-2.95 (2H, d).

5.k)8-hydroxyethyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

Intermediate 5.k is prepared according to a method similar to that ofStage 1.e, except for the fact that a molar proportion of 6/9 of BH₃relative to the substrate is used.

Mass spectrum: 274.3 MH+.

5.l)7-((1,1-dimethylethoxy)carbonyl)-8-hydroxyethyl-2-(2-methyoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

Intermediate 5.k (1.36 g; 5.0 mmol) is suspended in H₂O (5 ml) and amixture of di-t-butyldicarbonate (1.20 g; 5.5 mmol) in p-dioxane (10 ml)is added. The reaction medium is agitated vigorously and maintained atpH 8.0-8.4 by the dropwise addition of a 2.5N solution of NaOH until thereaction finishes (monitoring of the reaction by TLC on silica gel,eluant AcOEt:hexanes 3:2). The crude product is purified by flashchromatography on silica gel with an AcOEt:hexanes mixture 3:2 as eluant(Biotage system, pre-filled columns 4×15 cm). The fractions containingthe product are combined and concentrated under vacuum in order toproduce a white foam (1.60 g; 86%).

Mass spectrum: 374.3 MH+. NMR ¹H (DMSO-d6): 7.95-8.05 (1H, d,d);7.45-7.55 (1H, s); 7.10-7.25 (1H, m); 7.0-7.1 (1H, m); 6.9-7.05 (1H, m);5.05-5.15 (1H, t); 4.25-4.35 (1H, t); 4.05-4.2 (1H, broad s); 4.0-4.1(1H m); 3.9-4.0 (3H, s); 3.9-4.0 (1H, m); 3.25-3.35 (2H, m); 1.9-2.1(2H, m); 1.15-1.25 (9H, s).

5.m)7-((1,1-dimethylethoxy)carbonyl)-8-phenoxyethyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

Intermediate 5.1 (746 mg; 2.00 mmol) is dissolved in THF (10 ml)containing triphenylphosphine (550 mg; 2.1 mmol) and phenol (198 mg; 2.1mmol). The mixture is cooled down to 0° C. under nitrogen anddiethylazodicarboxylate (330 μl; 2.1 mmol) is added dropwise over 10minutes. The reaction mixture is then agitated for 2 hours at ambienttemperature. The reaction medium is then cooled down again to 0° C. andtriphenylphosphine (275 mg; 1.05 mmol) and phenol (99 mg; 1.05 mmol) areadded. Then diethylazodicarboxylate (166 μl; 1.05 mmol) is addeddropwise over 10 minutes then the mixture is agitated again for 1 hourat ambient temperature. The solvents are eliminated under reducedpressure and the crude product is purified by flash chromatography onsilica gel with an AcOEt:hexanes mixture 3:2 as eluant. The fractionscontaining the product are combined and concentrated under vacuum. Afterrecrystallization from AcOEt and hexanes, the desired product isobtained in the form of a white solid (863 mg; 96%).

Mass spectrum: 450.4 MH+.

5.n)7-(2-(((1,1-dimethylethoxy)carbonyl)amino)-1-oxo-3-((triphenylmethyl)thio)propyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine

Intermediate 5.m (850 mg; 1.89 mmol) is treated with a mixture of TFA(10 ml) containing iPr₃SiH (387 μl, 1.89 mmol) at ambient temperaturefor 20 min. The solvents are eliminated under reduced pressure and thecrude product is divided between AcOEt (15 ml) and a saturated solutionof NaHCO₃ (15 ml). The AcOEt phase is dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The deprotected product is coupledto Boc-(L)-Cys(Trt)-OH according to a method similar to that of Stage1.f (1.26 g; 84%).

5.o)7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-4,5,6,7-tetrahydro-imidazo-[1.2a]-piperazine

Product 5 is prepared starting from intermediate 5.n according to amethod similar to that of Stage 1.g.

Mass spectrum: 274.3 MH+. NMR ¹H (DMSO-d6 at 90° C.): 8.5-9.2 (3H, s,broad); 7.95-8.1 (1H, d); 7.85-8.0 (1H, s); 7.35-7.5 (1H, m); 7.15-7.35(3H, m); 7.0-7.15 (1H, t); 6.85-7.0 (3H, m); 5.9-6.1 (1H, s, broad);4.5-4.8 (2H, m, broad); 4.15-4.45 (3H, m, broad); 3.9-4.0 (3H, s);3.75-4.0 (1H, m, broad); 2.8-3.05 (2H, m, broad); 2.55-2.75 (2H, m,broad).

Example 67-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,dimer: 6

Compound 5.o (467 mg; 0.687 mmol) is solubilized in H₂O (25 ml) and thepH of the solution is adjusted to 7.2 by adding a dilute aqueoussolution of NH₄OH. Acetonitrile is added in order to produce a limpidsolution and the mixture is agitated at ambient temperature overnight.The crude product is purified by preparative HPLC on a C18 column usinga gradient of 15 to 40% CH₃CN in TFA at 0.1% over a period of 50minutes. The pure fractions of product are collected and lyophilized.The initial product is lyophilized twice from a dilute solution of HClin order to produce the product in the form of its hydrochloride (161mg; 45%).

Mass spectrum: 903.5 MH+. NMR ¹H (DMSO-d6 at 90° C.): 8.7-9.3 (3H, broads); 7.95-8.1 (1H, d); 7.85-8.0 (1H, s); 7.3-7.5 (1H, t); 7.1-7.3 (3H,m); 7.0-7.15 (1H, t); 6.8-7.0 (3H, m); 5.85-6.1 (1H, broad s); 4.7-4.9(1H, broad s); 4.45-4.7 (1H, broad m); 4.1-4.5 (4H, broad m); 3.85-4.0(4H, s); 3.3-3.5 (2H, broad m); 2.5-2.8 (2H, broad m).

Example 77-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine: 7

Compound 7 was prepared according to the synthesis diagram below:

7.p)7-((1,1-dimethylethoxy)carbonyl)-2-(2-methoxyphenyl)-8-(phenylthioethyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

Intermediate 5.1 (1.23 g; 3.30 mmol), tri-n-butylphosphine (1.64 ml;6.60 mmol) and phenyldisulphide (1.44 g; 6.60 mmol) are mixed in THF (10ml). The mixture is agitated at ambient temperature under argon for 4hours. The solvents are eliminated under reduced pressure and the crudeproduct is purified by flash chromatography on silica gel with anAcOEt:hexanes mixture 1:1 as eluant. The fractions containing theproduct are combined and concentrated under vacuum in order to producethe product in the form of a white foam (1.43 g; 93%).

Mass spectrum: 466.3 MH+.

7.q)7-((1,1-dimethylethoxy)carbonyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1,2-a]pyrazine

Intermediate 7.p (650 mg; 1.40 mmol) is dissolved in CH₂Cl₂ (10 ml) and3-chloroperoxybenzoic acid (483 mg; 2.80 mmol) is added in severalportions over a period of 10 minutes. The mixture is poured onto asilica column and eluted with a hexanes:AcOEt mixture 7:3, then ahexanes:AcOEt mixture 1:1 in order to produce the pure product (220 mg;32%).

Mass spectrum: 498.3 MH+. NMR ¹H (DMSO-d6 at 30° C.): 7.9-8.0 (3H, m);7.7-7.85 (1H, m); 7.6-7.75 (2H, m); 7.45-7.55 (1H,s); 7.15-7.25 (1H, m);6.9-7.1 (2H, m); 5.1-5.25 (1H, t); 4.1-4.3 (1H, broad d); 4.0-4.15 (1H,m); 3.8-4.0 (1H, m); 3.85-3.95 (3H, s); 3.6-3.8 (1H, m); 3.4-3.6 (1H,m); 3.2-3.4 (H₂O plus a blurred signal); 1.9-2.3 (2H,

Stage 7.n

Stage 7.n is carried out according to a method similar to Stage 5.n. Thecrude product is used without further purification in the followingstage.

Stage 7.o

The stage 7.o is carried out according to a method similar to Stage 5.o.

Mass spectrum: 501.3 MH+.

Preparation of the Compound of Formula (VII)

This compound, which is close to those described in Patent ApplicationPCT WO 97/30053, can be prepared according to the following synthesisdiagram:

Pharmacological Part

In order to illustrate the usefulness of the invention, there follows astudy on the effect of the treatment of a human MCF-7 cell line with thefollowing compounds:

-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine    disulphide, designated in this part as compound (I);-   bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl),    designated in this part as compound (II);-   7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,    designated in this part as compound (III);-   the compound of formula:    designated in this part as compound (IV);-   7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine,    designated in this part as compound (V);-   bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-2-(methoxyphenyl)-8-(1-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine],    designated in this part as compound (VI);-   the compound of formula:    designated in this part as compound (VII);    Procedures

Cell Line

The MCF-7 cell lines (human pleural cells, breast cancer) were acquiredfrom the American Tissue Culture Collection (Rockville, Md., USA).

Measurement of the intracellular quantity of cyclic AMP for the MCF-7cells

MCF-7 cells (2.10⁴ cells/well) seeded in 24-well plates are cultured for5 days in Dulbecco's modified Eagle medium (Gibco-Brl, Cergy-Pontoise,France) completed with 10% of foetal calf serum inactivated by heating(Gibco-Brl, Cergy-Pontoise, France), 50000 units/l of penicillin and 50mg/l streptomycin (Gibco-Brl, Cergy-Pontoise, France), and 2 mM ofglutamin (Gibco-Brl, Cergy-Pontoise, France). The culture medium isreplaced after two washes with a medium without a serum completed oruncompleted with the specified agents for a time indicated in thedifferent figures. Agents activating the production of cyclic AMP arethen added at 37° C. The reaction is stopped after 30 minutes bysuppressing the medium and rapidly adding 100 μl of a 0.1N solution ofHCl. These extracts are frozen at −80° C. until they are used. Theconcentration of cAMP is measured using a commercial measurement kit(reference NEK033 from NEN, Les Ulis, France), following themanufacturer's instructions. The radioactivity is determined by a Gammacounter (Gamma Master-1277, LKB, Turku, Finland).

Measurement of in Vitro Cell Proliferation

The MCF-7 cells (3000 cells/well) are cultured in 96-well plates in 80μl of Dulbecco's modified Eagle medium (Gibco-Brl, Cergy-Pontoise,France) completed with 10% of foetal calf serum inactivated by heating(Gibco-Brl, Cergy-Pontoise, France), 50000 units/l of penicillin and 50mg/l streptomycin (Gibco-Brl, Cergy-Pontoise, France), and 2 mM ofglutamin (Gibco-Brl, Cergy-Pontoise, France) were seeded on a 96-wellplate on day 0. The cells were treated on day 1 for 96 hours withincreasing concentrations of up to 50 μM of each of the compounds to betested. After this period, quantification of cell proliferation isdetermined by a colorimetric test, based on the cleavage of the WST1tetrazolum salt by the mitochondrial dehydrogenases in the viable cells,leading to the formation of formazan (Boehringer Mannheim, Meylan,France). These tests are carried out in duplicate with 8 determinationsper concentration tested. For each compound to be tested, the valuesincluded in the linear part of the sigmoid were retained for linearregression analysis and used to estimate the inhibitory concentration(IC₅₀).

Measurement of the MAP Kinase Activity

MCF7 cells (5.105 cells/well) are cultured in 6 wells in Dulbecco'smodified Eagle medium (Gibco-Brl, Cergy-Pontoise, France) completed with10% of foetal calf serum inactivated by heat (Gibco-Brl, Cergy-Pontoise,France), a mixture of antibiotics: 50000 units/l of penicillin and 50mg/l of streptomycin (Gibco-Brl, Cergy-Pontoise, France) and 2 mM ofglutamin (Gibco-Brl, Cergy-Pontoise, France). After 24 hours of culture,the cells are incubated for 48 hours in medium containing no serum inorder to return the cells to a state of rest. The cells are then treatedfor 1 hour either with compound I or with PD98059 (Calbiochem, FranceBiochem, Meudon, France), a specific inhibitor of MAP kinase activation.The cells are then stimulated (or not) for 5 minutes with 12.5 ng/ml ofepidermal growth factor (EGF). The reaction is stopped by two washeswith PBS (Gibco-Brl, Cergy-Pontoise, France), at 4° C. containingneither calcium nor magnesium and by adding 150 μl of lysis buffer at 4°C. the composition of which is the following: 10 mM of tris, 150 mM ofNaCl, 2 mM of EGTA, 2 mM of dithiothreitol, 1 mM of PMSF, 2 mM oforthovanadate, 10 μg/ml of leupeptin and 10 μg/ml of aprotinin.Measurement of the proteins contained in the extracts is carried out byBradford's method (Biorad reagents, Ivry-Sur-Seine, France). Theseextracts are frozen at −80° C. until they are used. The activity of theMAP kinase is measured using a commercial measurement kit (reference RPN84, Amersham Life Science, Les Ujis, France) following themanufacturer's instructions. The radioactivity is determined using aPackard scintillation counter (Tricarb 5000CA).

Equipment

The vasoactive intestinal peptide (VIP) was acquired from Bachem(Voisins le Bretonneux, France). The choleric toxin, forskolin,isoproterenol, prostaglandin E2 and PD 98059 were acquired fromCalbiochem (France Biochem, Meudon, France). The compounds of formulae(I), (II), (III), (IV), (V), (VI) and (VII) were supplied by BiomeasureInc. (Milford, Mass., USA). All these compounds were used followingtheir manufacturers' recommendations.

Results

FIG. 1 shows that activation of the adenylate cyclase by the cholerictoxin (200 ng/ml) or by forskolin (10 μM) leads to a very significantincrease in the cyclic AMP level. Pretreatment of the cells for 30minutes with 30 μM of comound (I) does not modify the production ofcyclic AMP induced by the direct activator of the adenylate cyclase,forskolin. On the other hand, the production of cyclic AMP stimulated bythe direct activator of the sub-unit, the choleric toxin, is greatlyinhibited by compound (I). This shows that the adenylate cyclase itselfis not modified by compound (I) and that the latter prevents theformation of the heterotrimeric complex.

VIP has been presented as an extra-cellular ligand of a receptor coupledwith the G protein which stimulates the synthesis of cyclic AMP in humanbreast cancer cells. FIG. 2 shows that treatment with VIP of MCF-7 humanbreast cancer cells increases the intracellular quantity of cyclic AMPin a concentration-dependent manner. A VIP concentration of 10 nM whichoffers a quasi-optimum production of cyclic AMP is used for thefollowing tests. This concentration agrees with the data alreadypublished relating to the T47D human breast cancer cell line.

FIG. 3 shows that a 30-minute pretreatment of the MCF-7 cells resultingfrom the in vitro cultures with the compound of formula (I) issufficient to inhibit the accumulation of cyclic AMP cyclic stimulatedby VIP in a concentration-dependent manner. An almost completeinhibition was obtained at a concentration of 100 μM of the compound offormula (I). These results show that a treatment with compound (I) issufficient to block the transduction of the signal the route of whichuses the heterotrimeric G proteins as mediators.

FIG. 4 shows that a treatment for an hour with the compound of formula(I) is sufficient to modify the response to VIP. Treatments of a longerduration (8 hours and 24 hours) continue to inhibit the production ofcyclic AMP but the main effect is obtained very rapidly.

Compound (I) is also capable of inhibiting the formation of cyclic AMPinduced by other agents which stimulate the receptors with seventransmembrane domains. In MCF7 cells, for example, the activity of theadenylate cyclase greatly increased by the prostaglandin E₂ is inhibitedby a treatment for 30 minutes with compound (I). This suggests thattreatment of the cells with compound (I) modifies the heterotrimericform of the G proteins by disassociating the sub-unit of the β/γ dimer.

Inhibition of stimulation by VIP is not restricted to compounds of astructure analogous to that of the compound of formula (I). As shown inTable I, compounds (II), (III), (IV), (V), (VI) and (VII) tested in thesame model are also capable of reducing the quantity of cyclic AMPinduced by VIP.

All these results suggest that the compounds tested modulate theactivity of the adenylate cyclase by modifying the heterotrimeric formof the G proteins. Now, it is known that the β/γ dimers can directlyactivate effectors leading to the activation of kinases regulated byextracellular signals (ERK's) or MAP kinases.

FIG. 6 shows that treatment of the cells for 1 hour with compound (I)doubles the basal activity of the MAP kinase. This suggests that bypreventing the formation of the heterotrimeric complex, compound (I)releases the heterodimer—which itself remains linked to the membrane andactivates the ras route. On the other hand, FIG. 7 shows that afterstimulation of the MAP kinase by the growth factor EGF for 5 minutes,the activity of the enzyme is increased by approximately 7 times.Pretreatment of the cells for 1 hour either with compound (I) or withPD98059, a specific inhibitor of MAP kinase activation, halves theactivity of the MAP kinase. These results suggest that compound (I)stimulates the basal state of the ras route and inhibits this same routeif it is stimulated, thus explainining its anti-proliferative effect.

Table II in fact shows that compounds (I), (II), (III) and (IV) arecapable of inhibiting the in vitro proliferation of MCF7 human tumourcells. TABLE I Compound Inhibition at 30 μM (I) 86% (II) 71% (III) 59%(IV) 52% (V) 68% (VI) 52% (VII) 65%Effects of Compounds I, II, III and IV Incubated for 30 Minutes on theProduction of Cyclic AMP Stimulated by VIP in MCF7 Cells.

The cells are incubated for 30 minutes in the presence or not ofcompounds I, II, III and IV (30 μM) which are then stimulated by 10⁻⁸ Mof VIP. The quantification of cyclic AMP is determined byradioimmunoassay. The data represents the average ±MSD (n=5 for thecontrol and n=1 for the different compounds ). TABLE II Compound testedIC₅₀ (μM) compound I 9.4 compound II 15.0 compound III 16.1 compound IV34.6Inhibition of the in Vitro Growth of MCF7 Cells by Compounds I, II, IIIand IV.

The results of the IC₅₀ are expressed in μM and represent the average of2 experiments.

1-12. (canceled)
 13. A method of treating a disorder selected fromoncogenesis, begnin and malign proliferative diseases in a patientcomprising administering to said patient an amount of a compound of theformula

wherein R₁ is selected from the group consisting of hydrogen, loweralkyl and lower alkylthio, R₂ and R₃ are individually hydrogen or loweralkyl, R₄ is

or ═O, R₅ is selected from the group consisting of a)hydrogen and b)lower alkyl, lower alkenyl, lower alkynyl, aryl, aryl lower alkyl,heterocycle and heterocycle lower alkyl, all unsubstituted orsubstituted with a member selected from the group consisting of loweralkyl, —O—R₁₀, —S(O)_(m)—R₁₀,

—NH—(SO₂)—R₁₁—COOR₁₂

m is an integer from 0 to 2, R₆ and R₇ are individually selected fromthe group consisting of a) hydrogen, b) unsubstituted

and c) lower alkyl, aryl, aryl lower alkyl, heterocycle and heterocyclelower alkyl, unsubstituted or substituted with a member of the groupconsisting of —OH, halogen, lower alkyl, lower alkoxy,

—COOH and

or taken together with the carbon atom to which they are attached formaryl or heterocycle, R₈ and R₉ are individually selected from the groupconsisting of a) hydrogen and b) lower alkyl, aryl lower alkyl,heterocycle and heterocycle lower alkyl, all unsubstituted orsubstituted with a member of the group consisting of —OH, halogen, loweralkyl, lower alkoxy,

taken together with the carbon atoms to which they are attached formaryl or heterocycle, R₁₀ and R₁ are individually selected from the groupconsisting of hydrogen, lower alkyl, aryl, aryl lower alkyl, heterocycleand heterocycle lower alkyl, R₁₃ is unsubstituted lower alkyl or loweralkyl substituted with a member selected from the group consisting ofaryl, aryl lower alkyl, heterocycle and heterocycle lower alkyl, R₁₃ isunsubstituted lower alkyl or lower alkyl substituted with a memberselected from the group consisting of lower alkyl, —O—R₁₀, —S(O)_(m)—R₁₀and

R₁₄ is hydrogen or lower alkyl, it being understood that Al can also bea dimmer when two R₁ are hydrogen, and its non-toxic, pharmaceuticallyacceptable salts, sufficient to treat said disorder.
 14. The method ofclaim 13 wherein the disorder is a begnin proliferative disease.
 15. Themethod of claim 13 wherein the disorder is a malign proliferativedisease.
 16. The method of claim 13 wherein the compound used isselected from the group consisting of7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methylphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-butyl-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-8-(1-methylpropyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;bis-1,1′-[7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclohexylmethyl)-2-(2-methoxyphenyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazinedisulphide;bis-1,1′-7-(2-amino-1-oxo-3-thiopropyl-(2-(1-naphthyl)-8-(2-methylpropyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazin-7-yl)disulphide;7-(2-amino-1-oxo-3-thioprpyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylmethoxy)methyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-2-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(1-phenylmethoxy)-ethyl-5,6,7,8-tetrahydroimidazo[1.2a-pyrazine;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydroimidazo[1.2a]pyrazine;7-(2-amino-1-oxo-3-thioproyl)-2-(2-methoxyphenyl)-8-(phenoxyethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine,or its dimeric form;7-(2-amino-1-oxo-3-thiopropyl)-2-(2-methoxyphenyl)-8-(phenylsulphonylethyl)-5,6,7,8-tetrahydro-imidazo[1.2a]pyrazine;and the non-toxic, pharmaceutically acceptable salts of the latter. 17.The method of claim 13 wherein the compound used is7-(2-amino-1-oxo-3-thiopropyl)-8-(cyclhexylmethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo[1.2a]pyrazineor a non-toxic, pharmaceutically acceptable salt of the latter.